Serum transferrin is a glycoprotein with a molecular weight of about 80 kD which comprises a single polypeptide chain with two N-linked polysaccharide chains. These polysaccharide chains are branched and terminate in five antennae with terminal sialic acid residues.
Wong and Regoeczi, in Int. J. Peptide Res. 9:241-248(1977), reported that human transferrin was naturally heterogeneous, occurring in variant forms with different levels of sialylation. In fact there appear to be six such variants, the pentasialo, tetrasialo, trisialo, disialo, monosialo and asialo transferrins.
In the normal healthy individual, the tetrasialo variant appears to predominate; however it has been found that the asialo, monosialo, disialo and, to some degree the trisialo variants occur in elevated levels in the blood of alcoholics (see van Eijk et al in Clin Chim Acta 132:167-171(1983), Stibler in Chin Chim 37:2029-2037(1991) and Stibler et al in "Carbohydrate-deficient transferrin (CDT) in serum as a marker of high alcohol consumption", Advances in the Biosciences, (Ed Nordmann et al), Pergamon, 1988, Vol. 71, pages 353-357).
The asialo, monosialo, disialo and trisialo variants are referred to herein as carbohydrate-deficient transferrin or CDT.
CDT has been found to be an effective marker for alcohol consumption, in particular for detecting and monitoring chronic alcohol consumption, and unlike conventional tests (e.g. GGT or MCV) can be used to screen for heavy alcohol intake in patients with liver disease.
As a result, several diagnostic assays for CDT have been described in patent and scientific literature.
In U.S. Pat. No. 4,626,355 (Joustra), Pharmacia AB describes a chromatographic assay in which a dilute serum sample is passed over an anionic ion exchange column with the pH and ion content of column and sample balanced to permit asialo, monosialo and disialo CDTs to eluate through in an isocratic procedure while the trisialo and the "normal" tetra and pentasialo variants are retained on the column. The CDT content of the eluate is then determined by competitive immobilization of CDT and radiolabelled-transferrin on an antibody carrying solid phase.
In a recent poster entitled "Rate nephelemetric determination of carbohydrate-deficient transferrin", Schellenberg, Martin, Benard, Circaud and Weill of Laboratoire de Biochimie CHU Trouseau, Tours, Centre Louis Sevestre, La Membrolle sur Choisille and Beckman France, Gagny, France described a similar isocratic chromatographic separation in which dilute serum is passed through an anionic ion exchange column causing the normal transferrin variants to be retained and allowing CDT to elute through. The eluate is then mixed with polyethyleneglycol and centrifuged, an anti-transferrin antibody is added to the supernatant and the CDT content is assessed by nephelometry.
Heil et al, in Anaesthetist 43:447-453(1994) have reported a further isocratic chromatographic procedure for CDT determination. In their procedure a dilute serum sample is passed through an anionic ion exchange resin, again causing normal transferrin variants to be retained while permitting transit of CDT. The CDT content of the eluate is determined by latex particle enhancement of CDT concentration in an immunoturbidimetric assay procedure.
In WO-91/19983 (Sundrehagen), Axis Research AS described an alternative CDT assay in which a dilute serum sample (in which the transferrin variants are bound by labelled (e.g. fluorophore-labelled) antibodies or antibody fragments reactive with all variants) is passed through an anionic ion exchange resin and the label concentration in the eluate is determined (e.g. by fluorescence measurement) as a function of eluate fraction. This assay relies upon the elution rates being different for the different variant:labelled binding partner complexes.
All of the assay procedures mentioned above rely upon relatively complex procedures which are not directly applicable to many of the automated multi-task diagnostic machines commonly used by diagnostic laboratories.